PROJECT SUMMARY/ABSTRACT Preterm birth represents a significant health burden worldwide, with particularly significant impact in the developing world. Influenza infection during pregnancy raises the risk of preterm birth four-fold, though the mechanism behind this remains mysterious. Recently, our laboratory showed that in vitro influenza infection elicits a more robust immune response in pregnant women than non-pregnant women, overturning the prevailing view that pregnancy is a generally immunosuppressed state. Upon examination of publicly available data, we also found that healthy pregnant women have a transcriptional signature consistent with acute influenza infection. These findings led to the central hypothesis that preterm birth is driven by excessive elevation of inflammatory pathways that are normally elevated during pregnancy, and that the magnitude of the response to influenza will predict the risk of preterm birth. However, no data yet exists on the intersection between preterm birth and influenza-associated inflammatory pathways, providing a rationale for the approach of performing perform holistic deep immune profiling on samples collected during pregnancy from Kenyan women who subsequently delivered preterm or at term. I will pursue my central hypothesis through two specific aims: 1) Defining how the cellular immune response to influenza during pregnancy differs between women who later deliver at term versus preterm, and 2) Identifying the transcriptional response to influenza in pregnant women who subsequently deliver at term or preterm. Under the first aim, a novel mass cytometry panel developed by the Blish lab will be used to study the cell subsets and immune functions at baseline and in response to in vitro influenza infection. This highly dimensional data will be analyzed manually and by unsupervised clustering algorithms that have successfully identified clinically relevant cell populations. Under the second aim, transcriptional arrays will be used to measure the levels of 6 million human gene transcripts, exon clusters and splice variants. This data will be used to identify genes that are significantly differentially expressed following in vitro influenza infection. This approach is innovative as it represents the first use of comprehensive immune profiling on a pregnancy cohort with known birth outcomes in a developing country. The proposed research is significant because it is expected to shed new light on the underlying pathogenesis of preterm birth, as well as point the way toward promising therapeutic targets. Ultimately, such knowledge has the potential shape the way pregnant women are assessed for preterm birth risk and provide therapeutic options for high-risk pregnancies.